Injectable polysaccharide-cell compositions

ABSTRACT

Slowly polymerizing polysaccharide hydrogels have been demonstrated to be useful as a means of delivering large numbers of isolated cells via injection. The gels promote engraftment and provide three dimensional templates for new cell growth. The resulting tissue is similar in composition and histology to naturally occurring tissue. This method can be used for a variety of reconstructive procedures, including custom molding of cell implants to reconstruct three dimensional tissue defects, as well as implantation of tissues generally.

BACKGROUND OF THE INVENTION

[0001] The present invention is generally in the area of creating newtissues using polysaccharide hydrogel-cell compositions.

[0002] Craniofacial contour deformities, whether traumatic, congenital,or aesthetic, currently require invasive surgical techniques forcorrection. Furthermore, deformities requiring augmentation oftennecessitate the use of alloplastic prostheses which suffer from problemsof infection and extrusion. A minimally invasive method of deliveringadditional autogenous cartilage or bone to the craniofacial skeletonwould minimize surgical trauma and eliminate the need for alloplasticprostheses. If one could transplant via injection and cause to engraftlarge numbers of isolated cells, one could augment the craniofacialosteo-cartilaginous skeleton with autogenous tissue, but withoutextensive surgery.

[0003] Unfortunately, attempts to inject dissociated cellssubcutaneously or to implant dissociated tissues within areas of thebody such as the peritoneum have not been successful. Cells arerelatively quickly removed, presumably by phagocytosis and cell death.

[0004] Cells can be implanted onto a polymeric matrix and implanted toform a cartilaginous structure, as described in U.S. Pat. No. 5,041,138to Vacanti, et al., but this requires surgical implantation of thematrix and shaping of the matrix prior to implantation to form a desiredanatomical structure.

[0005] Accordingly, it is an object of the present invention to providea method and compositions for injection of cells to form cellulartissues and cartilaginous structures.

[0006] It is a further object of the invention to provide compositionsto form cellular tissues and cartilaginous structures includingnon-cellular material which will degrade and be removed to leave tissueor cartilage that is histologically and chemically the same as naturallyproduced tissue or cartilage.

Summary of the Invention

[0007] Slowly polymerizing, biocompatible, biodegradable hydrogels havebeen demonstrated to be useful as a means of delivering large numbers ofisolated cells into a patient to create an organ equivalent or tissuesuch as cartilage. The gels promote engraftment and provide threedimensional templates for new cell growth. The resulting tissue issimilar in composition and histology to naturally occurring tissue. Inone embodiment, cells are suspended in a hydrogel solution and injecteddirectly into a site in a patient, where the hydrogel hardens into amatrix having cells dispersed therein. In a second embodiment, cells aresuspended in a hydrogel solution which is poured or injected into a moldhaving a desired anatomical shape, then hardened to form a matrix havingcells dispersed therein which can be be implanted into a patient.Ultimately, the hydrogel degrades, leaving only the resulting tissue.

[0008] This method can be used for a variety of reconstructiveprocedures, including custom molding of cell implants to reconstructthree dimensional tissue defects, as well as implantation of tissuesgenerally.

DETAILED DESCRIPTION OF THE INVENTION

[0009] Techniques of tissue engineering employing biocompatible polymerscaffolds hold promise as a means of creating alternatives to prostheticmaterials currently used in craniomaxillofacial surgery, as well asformation of organ equivalents to replaced diseased, defective, orinjured tissues. However, polymers used to create these scaffolds, suchas polylactic acid, polyorthoesters, and polyanhydrides, are difficultto mold and hydrophobic, resulting in poor cell attachment. Moreover,all manipulations of the polymers must be performed prior toimplantation of the polymeric material.

[0010] Calcium alginate and certain other polymers can form ionichydrogels which are malleable and can be used to encapsulate cells. Inthe preferred embodiment described herein, the hydrogel is produced bycross-linking the anionic salt of alginic acid, a carbohydrate polymerisolated from seaweed, with calcium cations, whose strength increaseswith either increasing concentrations of calcium ions or alginate. Thealginate solution is mixed with the cells to be implanted to form analginate suspension. Then, in one embodiment, the suspension is injecteddirectly into a patient prior to hardening of the suspension. Thesuspension then hardens over a short period of time. In a secondembodiment, the suspension is injected or poured into a mold, where ithardens to form a desired anatomical shape having cells dispersedtherein.

[0011] Polymeric Materials.

[0012] The polymeric material which is mixed with cells for implantationinto the body should form a hydrogel. A hydrogel is defined as asubstance formed when an organic polymer (natural or synthetic) iscross-linked via covalent, ionic, or hydrogen bonds to create athree-dimensional open-lattice structure which entraps water moleculesto form a gel. Examples of materials which can be used to form ahydrogel include polysaccharides such as alginate, polyphosphazines, andpolyacrylates, which are crosslinked ionically, or block copolymers suchas Pluronics™ or Tetronics™, polyethylene oxide-polypropylene glycolblock copolymers which are crosslinked by temperature or pH,respectively.

[0013] In general, these polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

[0014] Examples of polymers with basic side groups that can be reactedwith anions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

[0015] Alginate can be ionically cross-linked with divalent cations, inwater, at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

[0016] Polyphosphazenes are polymers with backbones consisting ofnitrogen and phosphorous separated by alternating single and doublebonds. Each phosphorous atom is covalently bonded to two side chains(“R”). The repeat unit in polyphosphazenes has the general structure(1):

[0017] where n is an integer.

[0018] The polyphosphazenes suitable for cross-linking have a majorityof side chain groups which are acidic and capable of forming saltbridges with di- or trivalent cations. Examples of preferred acidic sidegroups are carboxylic acid groups and sulfonic acid groups.Hydrolytically stable polyphosphazenes are formed of monomers havingcarboxylic acid side groups that are crosslinked by divalent ortrivalent cations such as Ca²⁺ or Al³⁺. Polymers can be synthesized thatdegrade by hydrolysis by incorporating monomers having imidazole, aminoacid ester, or glycerol side groups. For example, a polyanionicpoly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form hydrogel matrices.

[0019] Bioerodible polyphosphazines have at least two differing types ofside chains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl. The term bioerodible or biodegrable, as used herein, means apolymer that dissolves or degrades within a period that is acceptable inthe desired application (usually in vivo therapy), less than about fiveyears and most preferably less than about one year, once exposed to aphysiological solution of pH 6-8 having a temperature of between about25° C. and 38° C. Hydrolysis of the side chain results in erosion of thepolymer. Examples of hydrolyzing side chains are unsubstituted andsubstituted imidizoles and amino acid esters in which the group isbonded to the phosphorous atom through an amino linkage (polyphosphazenepolymers in which both R groups are attached in this manner are known aspolyaminophosphazenes). For polyimidazolephosphazenes, some of the “R”groups on the polyphosphazene backbone are imidazole rings, attached tophosphorous in the backbone through a ring nitrogen atom. Other “R”groups can be organic residues that do not participate in hydrolysis,such as methyl phenoxy groups or other groups shown in the scientificpaper of Allcock, et al., Macromolecule 10:824-830 (1977).

[0020] Methods for synthesis and the analysis of various types ofpolyphosphazenes are described by Allcock, H. R.; et al., Inorg. Chem.11, 2584 (1972); Allcock, et al., Macromolecules 16, 715 (1983);Allcock, et al., Macromolecules 19, 1508 (1986); Allcock, et al.,Biomaterials, 19, 500 (1988); Allcock, et al., Macromolecules 21, 1980(1988); Allcock, et al., Inorg. Chem. 21(2), 515-521 (1982); Allcock, etal., Macromolecules 22, 75 (1989); U.S. Pat. Nos. 4,440,921, 4,495,174and 4,880,622 to Allcock, et al.; U.S. Pat. No. 4,946,938 to Magill, etal.; and Grolleman, et al., J. Controlled Release 3, 143 (1986), theteachings of which are specifically incorporated herein by reference.

[0021] Methods for the synthesis of the other polymers described aboveare known to those skilled in the art. See, for example ConciseEncyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts,E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980). Manypolymers, such as poly(acrylic acid), are commercially available.

[0022] The water soluble polymer with charged side groups is crosslinkedby reacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃N⁺—\ / \ / \ /—⁺NR₃ can also be used.Aqueous solutions of the salts of these cations are added to thepolymers to form soft, highly swollen hydrogels and membranes. Thehigher the concentration of cation, or the higher the valence, thegreater the degree of cross-linking of the polymer. Concentrations fromas low as 0.005 M have been demonstrated to cross-link the polymer.Higher concentrations are limited by the solubility of the salt.

[0023] The preferred anions for cross-linking of the polymers to form ahydrogel are divalent and trivalent anions such as low molecular weightdicarboxylic acids, for example, terepthalic acid, sulfate ions andcarbonate ions. Aqueous solutions of the salts of these anions are addedto the polymers to form soft, highly swollen hydrogels and membranes, asdescribed with respect to cations.

[0024] A variety of polycations can be used to complex and therebystabilize the polymer hydrogel into a semi-permeable surface membrane.Examples of materials that can be used include polymers having basicreactive groups such as amine or imine groups, having a preferredmolecular weight between 3,000 and 100,000, such as polyethylenimine andpolylysine. These are commercially available. One polycation ispoly(L-lysine); examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan.

[0025] Polyanions that can be used to form a semi-permeable membrane byreaction with basic surface groups on the polymer hydrogel includepolymers and copolymers of acrylic acid, methacrylic acid, and otherderivatives of acrylic acid, polymers with pendant SO₃H groups such assulfonated polystyrene, and polystyrene with carboxylic acid groups.

[0026] Sources of Cells.

[0027] Cells can be obtained directed from a donor, from cell culture ofcells from a donor, or from established cell culture lines. In thepreferred embodiments, cells are obtained directly from a donor, washedand implanted directly in combination with the polymeric material. Thecells are cultured using techniques known to those skilled in the art oftissue culture.

[0028] Cell attachment and viability can be assessed using scanningelectron microscopy, histology, and quantitative assessment withradioisotopes. The function of the implanted cells can be determinedusing a combination of the above-techniques and functional assays. Forexample, in the case of hepatocytes, in vivo liver function studies canbe performed by placing a cannula into the recipient's common bile duct.Bile can then be collected in increments. Bile pigments can be analyzedby high pressure liquid chromatography looking for underivatizedtetrapyrroles or by thin layer chromatography after being converted toazodipyrroles by reaction with diazotized azodipyrrolesethylanthranilate either with or without treatment with P-glucuronidase.Diconjugated and monoconjugated bilirubin can also be determined by thinlayer chromatography after alkalinemethanolysis of conjugated bilepigments. In general, as the number of functioning transplantedhepatocytes increases, the levels of conjugated bilirubin will increase.Simple liver function tests can also be done on blood samples, such asalbumin production. Analogous organ function studies can be conductedusing techniques known to those skilled in the art, as required todetermine the extent of cell function after implantation. For example,islet cells of the pancreas may be delivered in a similar fashion tothat specifically used to implant hepatocytes, to achieve glucoseregulation by appropriate secretion of insulin to cure diabetes. Otherendocrine tissues can also be implanted. Studies using labelled glucoseas well as studies using protein assays can be performed to quantitatecell mass on the polymer scaffolds. These studies of cell mass can thenbe correlated with cell functional studies to determine what theappropriate cell mass is. In the case of chondrocytes, function isdefined as providing appropriate structural support for the surroundingattached tissues.

[0029] This technique can be used to provide multiple cell types,including genetically altered cells, within a three-dimensionalscaffolding for the efficient transfer of large number of cells and thepromotion of transplant engraftment for the purpose of creating a newtissue or tissue equivalent. It can also be used for immunoprotection ofcell transplants while a new tissue or tissue equivalent is growing byexcluding the host immune system.

[0030] Examples of cells which can be implanted as described hereininclude chondrocytes and other cells that form cartilage, osteoblastsand other cells that form bone, muscle cells, fibroblasts, and organcells. As used herein, “organ cells” includes hepatocytes, islet cells,cells of intestinal origin, cells derived from the kidney, and othercells acting primarily to synthesize and secret, or to metabolizematerials.

[0031] Addition of Biologically Active Materials to the hydrogel.

[0032] The polymeric matrix can be combined with humoral factors topromote cell transplantation and engraftment. For example, the polymericmatrix can be combined with angiogenic factors, antibiotics,antiinflammatories, growth factors, compounds which inducedifferentiation, and other factors which are known to those skilled inthe art of cell culture.

[0033] For example, humoral factors could be mixed in a slow-releaseform with the cell-alginate suspension prior to formation of implant ortransplantation. Alternatively, the hydrogel could be modified to bindhumoral factors or signal recognition sequences prior to combinationwith isolated cell suspension.

[0034] Methods of Implantation.

[0035] The techniques described herein can be used for delivery of manydifferent cell types to achieve different tissue structures. In thepreferred embodiment, the cells are mixed with the hydrogel solution andinjected directly into a site where it is desired to implant the cells,prior to hardening of the hydrogel. However, the matrix may also bemolded and implanted in one or more different areas of the body to suita particular application. This application is particlularly relevantwhere a specific structural design is desired or where the area intowhich the cells are to be implanted lacks specific structure or supportto facilitate growth and proliferation of the cells.

[0036] The site, or sites, where cells are to be implanted is determinedbased on individual need, as is the requisite number of cells. For cellshaving organ function, for example, hepatocytes or islet cells, themixture can be injected into the mesentery, subcutaneous tissue,retroperitoneum, properitoneal space, and intramuscular space. Forformation of cartilage, the cells are injected into the site wherecartilage formation is desired. One could also apply an external mold toshape the injected solution. Additionally, by controlling the rate ofpolymerization, it is possible to mold the cell-hydrogel injectedimplant like one would mold clay.

[0037] Alternatively, the mixture can be injected into a mold, thehydrogel allowed to harden, then the material implanted.

[0038] Specific Applications.

[0039] This technology can be used for a variety of purposes. Forexample, custom-molded cell implants can be used to reconstruct threedimensional tissue defects, e.g., molds of human ears could be createdand a chondrocyte-hydrogel replica could be fashioned and implanted toreconstruct a missing ear. Cells can also be transplanted in the form ofa thee-dimensional structure which could be delivered via injection.

[0040] The present invention will be further understood by reference tothe following non-limiting examples.

EXAMPLE 1 Preparation of a Calcium-Alginate-chondrocyte mixture andinjection into mice to form cartilaginous structures.

[0041] A calcium alginate mixture was obtained by combining calciumsulfate, a poorly soluble calcium salt, with a 1% sodium alginatedissolved in a 0.1 M potassium phosphate buffer solution (pH 7.4). Themixture remained in a liquid state at 4° C. for 30-45 min. Chondrocytesisolated from the articular surface of calf forelimbs were added to themixture to generate a final cellular density of 1×10⁷/ml (representingapproximately 10% of the cellular density of human juvenile articularcartilage).

[0042] The calcium alginate-chondrocyte mixture was injected through a22 gauge needle in 100 μl aliquots under the pannus cuniculus on thedorsum of nude mice.

[0043] The nude mice were examined 24 hours postoperatively, and allinjection sites were firm to palpation without apparent diffusion of themixture. Specimens were harvested after 12 weeks of in vivo incubation.On gross examination, the calcium alginate-chondrocyte specimensexhibited a pearly opalescence and were firm to palpation. The specimensweighed 0.11±0.01 gms (initial weight 0.10 gms). The specimens wereeasily dissected free of surrounding tissue and exhibited minimalinflammatory reaction. Histologically, the specimens were stained withhematoxylin and eosin and demonstrated lacunae within a basophilicground glass substance.

[0044] Control specimens of calcium alginate without chondrocytes had adoughy consistency 12 weeks after injection and had no histologicevidence of cartilage formation.

[0045] This study demonstrates that an injectable calcium alginatematrix can provide a three dimensional scaffold for the successfultransplantation and engraftment of chondrocytes. Chondrocytestransplanted in this manner form a volume of cartilage after 12 weeks ofin vivo incubation similar to that initially injected.

EXAMPLE 2 Effect of cell density on cartilage formation.

[0046] Varying numbers of chondrocytes isolated from the articularsurface of calf forelimbs were mixed with a 1.5% sodium alginatesolution to generate final cell densities of 0.0, 0.5, 1.0, and 5.0×10⁶chondrocytes/ml (approximately 0.0, 0.5, 1.0, and 5.0% of the cellulardensity of human juvenile articular cartilage). An aliquot of thechondrocyte-alginate solution was transferred to a circular mold 9 mm indiameter and allowed to polymerize at room temperature by the diffusionof a calcium chloride solution through a semi-permeable membrane at thebase of the mold. The gels formed discs measuring 2 mm in height and 9mm in diameter.

[0047] Discs of a fixed cellular density of 5×10⁶ cells/ml were alsoformed in which the concentration of the sodium alginate and themolarity of the calcium chloride solutions were varied.

[0048] All discs were placed into dorsal subcutaneous pockets in nudemice. Samples were harvested at 8 and 12 weeks and examined for grossand histological evidence of cartilage formation.

[0049] Examinations of 8 and 12 week specimens revealed that a minimumcell density of 5×10⁶ chondrocytes/ml was required for cartilageproduction which was observed only 12 weeks after implantation on grossexamination, the specimens were discoid in shape and weighed 0.13±0.01gms (initial weight 0.125 gms). The specimens were easily dissected freeof surrounding tissue and exhibited minimal inflammatory reaction.Histologically, the specimens were stained with hematoxylin and eosinand demonstrated lacunae within a basophilic ground glass substance.

[0050] Cartilage formation was independent of calcium chlorideconcentration used in gel polymerization. Cartilage was observed inspecimens with alginate concentrations varying from 0.5% to 4.0%;however, the lowest alginate concentration tested (0.5%) showed onlymicroscopic evidence of cartilage.

[0051] Cartilage can be grown in a subcutaneous pocket to apre-determined disc shape using calcium alginate gel as a support matrixin 12 weeks. Cartilage formation is not inhibited by eitherpolymerization with high calcium concentrations or the presence of highalginate concentrations but does require a minimum cellular density of5×10⁶ cells/ml.

[0052] The ability to create a calcium alginate-chondrocyte gel in agiven shape demonstrates that it is possible to use this technique tocustom design and grow cartilaginous scaffolds for craniofacialreconstruction. Such scaffolds have the potential to replace many of theprosthetic devices currently in use.

EXAMPLE 3 Preparation of Implantable Premolded Cell-polymer mixtures.

[0053] 250 μl aliquots of an isolated chondrocyte suspension was mixedwith 750 μls of a 2% (w/v) sodium alginate solution (0.1 M K₂HPO₄, 0.135M NaCl, pH 7.4). A 125 μl aliquot was placed into 9 mm diameter cellculture inserts with 0.45 μm pore size semipermeable membranes. Thecell-alginate mixture was placed into contact with a 30 mM CaCl₂ bathand allowed to polymerize for 90 minutes at 37° C. After 90 minutes, thecell-alginate gel constructs were removed from the mold and had adiameter of 9 mm and a height of 2 mm. The discs were placed into thewells of 24-well tissue culture plates and incubated at 37° C. in thepresence of 5% CO₂ with 0.5 ml of a solution containing Hamm's F-12culture media (Gibco, Grand Island, N.Y.) and 10% fetal calf serum(Gibco, Grand Island, N.Y.) with L-glutamine (292 μg/ml), penicillin(100 U/ml), streptomycin (100 μg/ml) and ascorbic acid (5 μg/ml) for 48hrs.

[0054] Using this method, bovine chondrocyte-alginate discs wereprepared, then implanted in dorsal subcutaneous pockets in athymic miceusing standard sterile technique. After one, two, and three months,athymic mice were sacrificed, and the gel/chondrocyte constructsremoved, weighed and placed in appropriate fixative. The cell-polymercomplexes were studied by histochemical analysis.

[0055] Cartilage formation was observed histologically after threemonths of in vivo incubation at an initial chondrocyte density of 5×10⁶cell/ml.

[0056] The above protocol was modified by using a range of CaCl₂concentration and a range of sodium alginate concentrations. Cartilageformation was observed using 15, 20, 30, and 100 mM CaCl₂ baths and 0.5,1.0, 1.5, 2.0, and 4.0% sodium alginate solutions.

[0057] By changing the mold within which the cell-alginate construct iscreated, the shape of the implant can be customized. Additionally, themold need not be semipermeable as calcium ion can be directly mixed withthe cell-alginate solution prior to being placed within a mold. The keyfeature is that the construct can be fashioned into a given shape priorto implantation.

EXAMPLE 4 Preparation of injectable osteoblasts-hydrogel mixtures.

[0058] Using the methodology described above, bovine osteoblasts havebeen substituted for chondrocytes and injected into animals using ahydrogel matrix.

[0059] Histology after 12 weeks of in vivo incubation showed thepresence of early bone formation.

EXAMPLE 5 Use of the hydrogel matrix to form an immunoprotective matrixaround the implanted cells.

[0060] By fashioning a cell-alginate construct as described above, onecan use the hydrogel matrix to sterically isolate the encapsulated cellsfrom the host immune system, and thereby allow allogenic celltransplants to form new tissues or organs without immunosuppression.

[0061] Bovine chondrocytes in an alginate suspension were transplantedinto normal immune-competent mice. Histology after six weeks of in vivoincubation shows the presence of cartilage formation. Gross examinationof the specimens does not demonstrate features of cartilage. Literaturestates that similar chondrocyte xenografts without alginate do not formcartilage.

[0062] Modifications and variations of the compositions and methods ofthe present invention will be obvious to those skilled in the art fromthe foregoing detailed description. Such modifications and variationsare intended to come within the scope of the following claims.

We claim:
 1. A method for implanting tissue into an animal comprisingmixing a biodegradable, biocompatible hydrogel solution with dissociatedcells and implanting the mixture into the animal.
 2. The method of claim1 wherein the hydrogel solution is hardened prior to implantation in theanimal.
 3. The method of claim 1 wherein the hydrogel is injected intothe animal as a cell suspension, which then hardens.
 4. The method ofclaim 1 wherein the hydrogel is selected from the group consisting ofalginate, polyphosphazines, polyethylene oxide-polypropylene glycolblock copolymers, poly(acrylic acids), poly(methacrylic acids),copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate),and sulfonated polymers.
 5. The method of claim 4 wherein the hydrogelis hardened by exposure to an agent selected from the group consistingof ions, pH changes, and temperature changes.
 6. The method of claim 5wherein the hydrogel is hardened by interaction with ions selected fromthe group consisting of cations selected from the group consisting ofcopper, calcium, aluminum, magnesium, strontium, barium, tin, and di-,tri- or tetra-functional organic cations; anions selected from the groupconsisting of low molecular weight dicarboxylic acids, sulfate ions andcarbonate ions.
 7. The method of claim 4 wherein the hydrogel is furtherstabilized by cross-linking with a polyion.
 8. The method of claim 1wherein the cells are selected from the group consisting of chondrocytesand other cells that form cartilage, osteoblasts and other cells thatform bone, muscle cells, fibroblasts, and organ cells.
 9. The method ofclaim 1 wherein the hydrogel is molded to form a specific shape prior toimplantation.
 10. The method of claim 1 wherein the hydrogel is moldedto form a specific shape after mixing with the cells and being implantedinto the animal.
 11. A composition for implanting tissue into an animalcomprising a hydrogel solution mixed with dissociated cells.
 12. Thecomposition of claim 11 wherein the hydrogel solution is hardened priorto implantation in the animal.
 13. The composition of claim 11 whereinthe hydrogel is injected into the animal as a cell suspension, whichthen hardens.
 14. The composition of claim 11 wherein the hydrogel isselected from the group consisting of alginate, polyphosphazines,polyethylene oxide-polypropylene glycol block copolymers, poly(acrylicacids), poly(methacrylic acids), copolymers of acrylic acid andmethacrylic acid, poly(vinyl acetate), and sulfonated polymers.
 15. Thecomposition of claim 14 wherein the hydrogel is hardened by exposure toan agent selected from the group consisting of ions, pH changes, andtemperature changes.
 16. The composition of claim 15 wherein thehydrogel is hardened by interaction with ions selected from the groupconsisting of cations selected from the group consisting of copper,calcium, aluminum, magnesium, strontium, barium, tin, and di-, tri- ortetra-functional organic cations; anions selected from the groupconsisting of low molecular weight dicarboxylic acids, sulfate ions andcarbonate ions.
 17. The composition of claim 14 wherein the hydrogel isfurther stabilized by cross-linking with a polyion.
 18. The compositionof claim 11 wherein the cells are selected from the group consisting ofchondrocytes and other cells that form cartilage, osteoblasts and othercells that form bone, muscle cells, fibroblasts, and organ cells.